This invention relates to avian vaccines, and more particularly to avian vaccines derived from avian pneumoviruses.
Avian pneumovirus (xe2x80x9cAPVxe2x80x9d) is a member of the Paramyxoviridae family of viruses. Pringle, Arch. Virol. 141:2251-2256 (1996). It is the etiological agent of turkey rhinotracheitis, causing an acute upper respiratory tract infection characterized by coughing, nasal discharge, tracheal rales, foamy conjunctivitis and sinusitis in young poults. In laying birds, there is transient drop in egg production along with mild respiratory tract illness. Jones, Avian Pathol., 25:639-648 (1996). While uncomplicated cases of APV infection usually result in low mortality, secondary bacterial and/or viral infections can result in up to 25% mortality. Id.
APV was first detected in South Africa in 1978, and later diagnosed in the UK, France, Spain, Germany, Italy, Netherlands, Israel, and Asia. Alexander, In Diseases of Poultry; 10th edition, Barnes et al., (eds.), 541-569 (1997); and Jones, supra. The first United States APV case was in Colorado in 1996. Kleven, Proc. U.S. Animal Health. Assoc. 101st Annual Mtg., 486-491 (1997). Subsequent APV infections were reported in Minnesota and neighboring states. Lauer, Minnesota Poultry Testing Laboratory Monthly Report, (1999). By 1999, at least 37% of the turkey flocks in Minnesota were positive for APV antibodies, causing economic losses of approximately 15 million dollars.
The tremendous economic pressure caused by APV outbreaks has caused some farmers to expose young turkey flocks to homogenized lungs obtained from APV infected turkeys in a desperate attempt to immunize young poults. These drastic steps taken by farmers are not safe and effective methods for protecting turkeys from APV infection. Thus, there exists a need for safe and effective vaccines against APV infections in birds including turkeys.
In one aspect, the invention features a composition includes an immunologically effective amount of an attenuated avian pneumovirus. In one embodiment, the attenuated avian pneumovirus is sequestered. In some embodiments, the composition further includes an acceptable pharmaceutical carrier. In other embodiments, the attenuated avian pneumovirus is p41.
These compositions containing immunologically effective amounts of attenuated avian pneumoviruses are effective for lowering the risk of an avian pneumovirus infection in wild birds and domesticated birds. In particular, the compositions are useful for preventing an avian pneumovirus infection in poultry including turkeys, chickens, ducks, geese, pheasants, partridges, guinea fowl, peacocks. In addition, the APV compositions are effective for ameliorating of the clinical signs of an avian pneumovirus infection in a challenged bird.
In another aspect, the inventions features methods for preparing an attenuated avian pneumovirus composition by infecting or inoculating a cell culture with an avian pneumovirus, and serially propagating the infected cell culture until the avian pneumovirus becomes attenuated. In some embodiments, methods for preparing an attenuated avian pneumovirus further include the step of removing the attenuated avian pneumovirus from the infected cell culture. The cell cultures can be avian or non-avian cell cultures, or a combination thereof in any order. For example, the cell cultures can include vero cells, QT-35 cells or CEF cells. In some embodiments, the avian pneumovirus is selected from the group consisting of the European A, European B, Colorado, Minnesota 1A, Minnesota 1B, Minnesota 2A, and Minnesota 2b isolates.
In some embodiments, the infected cell culture is serially propagated at least 20 times, at least 40 times, at least 60 times, or at least 100 times, or any number of passages between 10 and 110 passages. For example, in one embodiment the infected cell culture is serially propagated 41 times.
These methods are effective for producing an attenuated avian pneumovirus that is effective for reducing or preventing the incidence of the clinical signs of an avian pneumovirus infection in poultry and, in particular, turkeys and chickens.
In another aspect, the invention features a method for preparing an attenuated avian pneumovirus composition that includes the steps of inoculating or infecting an avian cell culture with an avian pneumovirus, propagating the avian pneumovirus in the avian cell culture, inoculating or infecting a non-avian cell culture with an avian pneumovirus isolated from the propagated avian cell culture, propagating the non-avian infected cell culture until the avian pneumovirus becomes attenuated, and isolating the attenuated avian pneumovirus from the non-avian infected cell culture.
In another aspect, the invention features a method for reducing the risk of an avian pneumovirus infection in a bird by inoculating a bird with an immunologically effective amount of an attenuated avian pneumovirus composition. In some embodiments, the inoculated bird is allowed to become seropositive. Although many different dosages may be used, particularly useful dosages include inoculating a bird with at least 1.6xc3x97106 TCID50 of the attenuated avian pneumovirus composition, at least 1xc3x97102 TCID50 of the attenuated avian pneumovirus composition, or at least 1xc3x97101 TCID50 of the attenuated avian pneumovirus composition.
Any method of inoculation can be used including applying the composition to one or more eyes of a bird and/or one or more nostril of a bird, or perhaps supplying the attenuated avian pneumovirus composition in the drinking water of a bird. Inoculated birds can be members of a flock of birds and the inoculated or vaccinated birds can cause a majority of the flock to become seropositive. In some embodiments, the method is effective for reducing the incidence of the clinical signs of an avian pneumovirus infection in a challenged bird.
In another aspect, the invention features, an inoculated bird, which is a bird containing an inoculant of an immunologically effective amount of an isolated attenuated avian pneumovirus. In some embodiments, the bird is allowed to become or is seropositive for avian pneumovirus. In another aspect, the invention features a body part, such as a meat portion, of an inoculated or vaccinated bird. In particular, these birds can be turkeys.
In yet a further aspect, the invention features compositions containing immunologically effective amounts of inactivated avian pneumovirus. In some embodiments, the composition further includes an acceptable pharmaceutical carrier or adjuvant. In other embodiments, the inactivated avian pneumovirus is an inactivated form of an attenuated avian pneumovirus such as an inactivated form of p41. Compositions containing inactivated avian pneumoviruses are also effective for lowering the risk of an avian pneumovirus infection in poultry, such as chickens or turkeys, and in other domesticated and wild birds. The composition are also effective for ameliorating the clinical signs of an avian pneumovirus infection in a challenged bird. In some embodiments, the avian pneumovirus is a formalin or xcex2-propiolactone inactivated avian pneumovirus.
In another embodiment, the invention features a composition containing an immunologically effective amount of an isolated attenuated avian pneumovirus wherein the attenuated avian pneumovirus became attenuated by propagating an avian pneumovirus in a non-avian host, such as a vero cell. In other embodiments, the composition containing an immunologically effective amount of an isolated attenuated avian pneumovirus was serially propagated in an avian host before being propagated in a non-avian host in vitro.
In another embodiment, a method for preparing an attenuated avian pneumovirus composition includes the steps of infecting a host with an avian pneumovirus, propagating the avian pneumovirus in the host, infecting a cell culture with an avian pneumovirus isolated from the propagated host, propagating the infected cell culture until the avian pneumovirus becomes attenuated, and perhaps isolating the attenuated avian pneumovirus from the non-avian infected cell culture. Useful hosts for such a method include embryonated chicken eggs, embryonated turkey eggs, and tracheal organ cultures. Useful cell cultures for such a method include chicken embryo fibroblast, quail tumor cell lines, and vero cells.
In another aspect, the invention features an article of manufacture containing attenuated or inactivated APV compositions. The compositions can be combined with packaging materials and instructions for their use. The articles of manufacture may combine one or more isolated attenuated or inactivated APV vaccines. In addition, the articles of manufacture may further include antibodies, indicator molecules, and/or other useful agents for detecting other avian diseases together with isolated attenuated APV vaccines. The instructions can describe how an isolated attenuated or inactivated APV vaccine is effective for preventing the incidence of an APV infection, preventing the occurrence of the clinical signs of an APV infection, ameliorating the clinical signs of an APV infection, lowering the risk of the clinical signs of an APV infection, lowering the occurrence of the clinical signs of an APV infection and/or spread of APV infections in birds.
Articles of manufacture can also include diagnostic molecules that are effective for detecting the presence of APV or other avian infections in birds. Moreover, it is to be understood that isolated attenuated or inactivated APV vaccines themselves and/or articles of manufacture that include isolated attenuated or inactivated APV vaccines can include other components conventional to the art, for example sterile water, pharmaceutical carriers, vaccine carriers, and buffers that are useful for maintaining the viability of the APV vaccines. The APV vaccines and/or articles of manufacture may also contain other attenuated or inactivated virus strains, microorganisms, and antigens that protect the inoculated birds against other avian diseases. Conveniently the isolated attenuated APV vaccines may be provided in a pre-packaged form in quantities sufficient for a protective dose for a single bird or for a pre-specified number of birds in, for example, sealed ampoules, capsules or cartridges.
It is to further understood that each of the embodiments may be combined with any of the other embodiments described herein. For example, the methods disclosed herein are useful with any of the compositions, or articles of manufacture, and the dosages may be used in any of the various embodiments described herein.
Advantages of the invention include a safe and effective way to protect birds, especially turkeys, from an APV infection. Moreover, the invention can provide methods for raising antibodies to APV to be used in diagnostic kits and may be used in the diagnostic kits for detecting the presence of APV.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. Amino acid designations may include full name, three letter, or single letter designations as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.